Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

An acetone extract, chloroform extract or hexane extract of Angelicae
sinensis and/or the active components purified therefrom, such as
n-butylidenephthalide, are administered alone or in combination with one
or more chemotherapy drugs and are effective in treating cancers.

8. The method of claim 5, wherein the n-butylidenephthalide is
administered via an oral, parenteral, or intravenous route.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This is a divisional application of U.S. patent application Ser. No.
11/561,713, filed Nov. 20, 2006, which was a divisional application of
U.S. patent application Ser. No. 11/246,009, filed Oct. 7, 2005, which
claims the benefit of U.S. Provisional Application No. 60/616,636, filed
Oct. 8, 2004, the entire disclosures of each of which are hereby
incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002]The invention mainly relates to a new use of an acetone extract,
chloroform extract or hexane extract of Angelicae sinensis and the active
components purified therefrom in the treatment of cancers.

[0003]Cancers are abnormal cell proliferations that result from the
accumulation of genetic changes in cells endowed with proliferative
potential. Treatment of cancers has relied mainly on surgery,
chemotherapy, radiotherapy and more recently immunotherapy. However, new
approaches for treating and preventing cancers are still desired.

[0005]CN1053747 disclosed that Angelicae sinensis (Oliv) Diels, ASD, and
the ASDP and ASDE as effective components of an adjuvant was prepared,
and could be used as an immunological adjuvant to genetically-engineered
hepatitis B vaccines. It was reported in CN1109356 that the effective
component, lactones (ASDE), extracted from Angelicae sinensis (oliv)
diels, ASD, could be used as an immunological adjuvant, which can enhance
the immunogenicity and help lower toxicity. Kumazawa et al. provided
immunostimulating polysaccharides separated from a hot water extract of
Angelicae sinensis, which could be used as a potent adjuvant for its
anti-tumor activity as observed in the prolongation of the survival
period of mice bearing Ehrlich ascites cells (Y. Kumazawa, et al.,
Immunology, Vol. 47, p. 75, 1982). However, this prior art reference
provides only a general description of the treatment of cancers with the
polysaccharides separated from Angelicae sinensis through their
immunostimulating activity, without sufficient evidence regarding the
mechanism.

BRIEF SUMMARY OF THE INVENTION

[0006]This invention provides that an acetone extract, chloroform extract
or hexane extract of Angelicae sinensis, or at least one component
purified therefrom, such as n-butylidenephthalide (BP), can also inhibit
telomerase activity of cancer cells and further 10 induce their apoptosis
so that they can be used to treat malignant neoplasms. Therefore, an
acetone extract, chloroform extract or hexane extract of Angelicae
sinensis, and the components purified therefrom, such as
n-butylidenephthalide, are potent for manufacturing of medicines for the
treatment of cancers, and can be used in combination with chemotherapy
drugs through their activities on cell cycle regulation, and telomerase
inhibition.

[0007]Accordingly, one object of the present invention is to provide a
method for inhibiting cancer cell proliferation and migration in tumor
tissues.

[0008]Another object of the present invention is to provide a method for
inhibiting telomerase activity of cancer cells.

[0009]Yet another object of the present invention is to provide a method
for inducing apoptosis of cancer cells.

[0010]Another object of the present invention is to provide the use of an
acetone extract, chloroform extract or hexane extract of Angelicae
sinensis, or at least one component purified therefrom, such as
n-butylidenephthalide, for manufacturing medicine for the treatment of
cancer, and as an adjuvant in combination with chemotherapy drugs through
their activities on cell cycle regulation and/or telomerase inhibition.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0011]The foregoing summary, as well as the following detailed description
of the invention, will be better understood when read in conjunction with
the appended drawings 30 relating to embodiments which are presently
preferred. It should be understood, however, that the invention is not
limited to the precise embodiments shown.

[0012]In the drawings:

[0013]FIG. 1a provides the results of the cell cycle analysis, which
demonstrates that treatment with 70 μg/ml AS-C (the chloroform extract
of Angelicae sinensis) enhanced cell cycle accumulation at G0/G1 phase
(>90%) in GBM cells (DBTRG-05M) (*p<0.05) with a concurrent
decrease in S phase. The results on G5T/VGH were about the same and not
shown in the graph.

[0019]FIG. 5 shows that the survival rate of the AS-C treated mice
(dose--500 mg/kg) was significantly prolonged as compared with the
control group (p<0.0001), wherein the DBTRG-05MG cell line was used.

[0021]FIG. 7 shows the inhibitory effect of the AS-C treatment (500 mg/kg
by intra-peritoneal or subcutaneous administration) on the xenograft
tumor growth of mice (p<0.005), wherein the DBTRG-05MG cell line was
used.

[0022]FIG. 8 shows the inhibitory effect of the BP treatment (300 mg/kg)
on tumor volume of GBM in situ (RG2) on rats, which was calculated with
MRI imaging using echo-planar imaging capability (* p<0.05, **
p<0.001).

[0023]FIG. 9 shows the inhibitory effect of BP treatment, at different
dosages (70 to 800 mg/kg), on xenograft tumor growth on mice
(p<0.005), wherein the DBTRG-05MG cell line was used.

[0024]FIG. 10 shows the effect of BP treatment (70 to 800 mg/kg) on the
prolongation of survival period of nude mice with xenograft tumor
(subcutaneous DBTRG-05MG) (p<0.001).

DETAILED DESCRIPTION OF THE INVENTION

[0025]This invention provides that the organic solvent extracts of
Angelicae sinensis, or the components purified therefrom, such as
n-butylidenephthalide (BP), can inhibit telomerase activity of cancer
cells and further induce their apoptosis. Therefore, they can inhibit
cancer cell proliferation and can be used to treat cancers.

[0026]Preparation of Angelicae Sinensis Extracts

[0027]Angelicae sinensis (Dangqui) has long been used in blood diseases
and female diseases. Normally, the dried root of Angelica sinensis
(Oliv.) Diels, belonging to the family of Umbelliferae, is used. Angelica
sinensis (AS) is appreciated by those skilled in this art. A variety of
techniques are well known in the art for extracting, separating, and/or
purifying individual active components of Angelicae sinensis. The organic
solvent extracts of Angelicae sinensis may be obtained by any standard
procedures commonly used in the field. According to the invention,
Angelicae sinensis is extracted with acetone, chloroform, or hexanes. In
one embodiment of the invention, the dried and powdered rhizomes of
Angelicae sinensis were extracted with acetone as a solvent to give an
extract as AS-A. Furthermore, AS-A was further extracted with chloroform
to give an extract as AS-C; and AS-A was further extracted with hexanes
to give an extract as AS-H.

[0028]Purification of Active Components

[0029]Active components of Angelicae sinensis may be isolated and/or
purified from the organic solvent extracts of Angelicae sinensis by using
any techniques known in the art. The active components may be purified
from Angelicae sinensis in any form, particularly the rhizomes. Various
techniques that may be employed in the further purification include
filtration, selective precipitation, extraction with organic solvents,
extraction with aqueous solvents, column chromatography, high performance
liquid chromatography (HPLC), etc. According to the invention, some
active components were purified from the organic solvent extracts of
Angelicae sinensis, such as ligustilide and n-butylidenephthalide, which
can induce tumor cell apoptosis. In one embodiment of the invention, E-
and Z-geometrical isomers of n-butylidenephthalide (BP) were separated
with column chromatography and characterized with HPLC and NMR.

[0030]Mechanisms of Cancer Treatment

[0031]Telomeres, the extremities of eukaryotic chromosomes, are essential
for maintaining the integrity of the genome and are a key determinant of
cellular aging and immortality (N. W. Kim, M. A. Piatyszek, K. R. Prowse,
C. B. Harley, M. D. West, P. L. C. Ho, G. M. Coviello, W. E. Wright, S.
L. Weinrich, J. W. Shay, Science, 266, 2011-2015 (1994)). Telomere length
and the rate of its reduction vary among organs and individuals. Large
interchromosomal variation in telomere length exists in mice and humans
and an aberration of a telomere in a single chromosome can lead to
abnormal chromosomal segregation (L. L. Sandell, V. A. Zakian, Cell, 75,
729-739 (1993)). Therefore, it is concluded that the regulation and
maintenance of telomere length variation play an important role in cancer
development. Apparently cells have a system to protect against both
critical shortening and abnormal elongation of the telomere. Telomerase
has been identified as one of the telomere length regulators (G. B.
Morin, Cell, 59, 521-529 (1989)). Hence, any compound or substance having
a selective inhibiting telomerase activity can inhibit tumor cell growth
and thus further induce cell apoptosis of tumor cells.

[0032]Apoptosis is another mechanism of cancer therapy, which has become
one of the newest areas of cell biology research.

[0033]The activation of the apoptosis program is regulated by various
signals from both intracellular and extracellular stimuli. Indeed, in
recent years evidence is beginning to accumulate that many (and perhaps
all) agents of cancer chemotherapy kill tumour cells by launching the
mechanisms of apoptosis. New drugs associated with apoptosis are expected
to be most effective against tumours with high proliferation rates. Many
such candidates are being screened for use in the treatment of cancer
(Ricardo Perez-Tomas, Beatriz Montaner, Esther Llagostera, Vanessa
Soto-Cerrato, Biochemical Pharmacology, 66, 1447-1452 (2003)).

[0034]Apoptosis is monitored by the analysis of two commonly used
endpoints--the morphological changes of cells (condensation of nuclear
chromatin, formation of apoptotic bodies) and DNA fragmentation into
large fragments (300 and 50 kbp) and then to oligonucleosomesized
fragments (multiples of 200 bp), which appear as a "ladder" of DNA bands
upon agarose gel electrophoresis. Although observation of these endpoints
is an indicator of apoptosis, quantification of the percentage of
apoptotic cells in a population by such an assay is impossible. For this
purpose, we also used the TUNEL assay during which
fluorescently-biotinylated nucleotides were added to the ends of DNA
fragments within fixed cells (Jacques Piettea, Cedric Volantia, Annelies
Vantieghemb, Jean-Yves Yvette Habrakena, Patrizia Agostinis, Biochemical
Pharmacology, 66, 1651-1659 (2003)).

[0035]The relative contribution of the receptor and the mitochondrial
pathways to drug-induced apoptosis has been a subject of controversy. It
depends on the type of the cytotoxic drug itself, the dose and kinetics
or on differences between certain cell types, which affects the cell type
dependent signaling in the Fas/FasL pathway.

[0037]It has been reported in the literature that acetone extract,
chloroform extract or hexane extract of Angelicae sinensis and its active
components have anti-angina, anti-agglutination and certain other
activities on the cardiovascular system.

[0038]Surprisingly, we found in this invention that the acetone extract,
chloroform extract or hexane extract of Angelicae sinensis, and the
active components purified therefrom, such as n-butylidenephthalide, have
anti-cancer activities.

[0039]According to this invention, the growth of several cancer cell lines
were tested against the acetone extract, chloroform extract or hexane
extract of Angelicae sinensis, the active components purified therefrom,
such as n-butylidenephthalide. It was found that they were cytotoxic to
cancer cells; they inhibited telomerase activity of cancer cells (as
shown in Example 7); they suppressed cancer cell proliferation (as shown
in Example 2) and they also induced cancer cell apoptosis (as shown in
Examples 3 and 4). Furthermore, animal studies also showed they were
effective in suppressing cancer growth (as shown in Examples 5 and 6).
They are therefore potent for treating cancers, particularly human
malignant glioblastoma, colorectal cancer, leukemia, neuroblastoma,
hepatoma, breast, ovarian and lung cancers.

[0040]Pharmaceutical Compositions

[0041]The acetone extract, chloroform extract or hexane extract of
Angelicae sinensis, active components purified therefrom, and the
derivatives according to the present invention may be administered by any
conventional route of administration including, but not limited to, oral,
parenteral, intraperitoneal (ip), intravenous (iv), intramuscular (im),
subcutaneous (sc), pulmonary, transdermal, buccal, nasal, sublingual,
ocular, rectal, vaginal or other routes. It will be readily apparent to
those skilled in the art that any dosage or frequency of administration
that provides the desired therapeutic effect is suitable for use in the
present invention. In a preferred embodiment of the invention, they are
administered by oral delivery, using methods known to those skilled in
the art of drug or food delivery.

[0042]For the purposes of therapeutic administration, the acetone extract,
chloroform extract or hexane extract of Angelicae sinensis, active
components purified therefrom, and the derivatives may be in the form of
a tablet, pill, capsule, granule, gel, powder, sterile parenteral
solution or suspension, metered aerosol or liquid spray, or suppository,
depending on the administration route. To prepare a pharmaceutical
composition of the present invention, the organic solvent extracts of
Angelicae sinensis, active components purified therefrom, or the
derivatives are admixed with a pharmaceutically acceptable carrier
according to conventional pharmaceutical compounding techniques, wherein
the carrier may take a wide variety of forms depending on the form of
preparation desired for administration. Suitable pharmaceutically
acceptable carriers are well known in the art. Descriptions of some
pharmaceutically acceptable carriers may be found in The Hand Book of
Pharmaceutical Excipients, published by the American Pharmaceutical
Association and the Pharmaceutical Society of Great Britain. For
instance, the tablets, capsules, gels, solutions or suspensions may also
include the following components: a pharmaceutically acceptable excipient
or carrier, which is a non-toxic, inert solid or semi-solid, diluent,
encapsulating material, a gel base or formulation auxiliary of any type.
The solutions and suspensions may contain auxiliaries, such as water for
injection, saline solution, polyethylene glycols, glycerin, propylene
glycol or other synthetic solvents; proteins such as serum albumin to
enhance solubility; antibacterial agents such as benzyl alcohol or methyl
parabens; antioxidants such as ascorbic acid or sodium bisulfite; buffers
such as acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. The solution or suspension
preparations can be enclosed in ampoules, disposable syringes or multiple
dose vials made of glass or plastic.

[0043]The acetone extract, chloroform extract or hexane extract of
Angelicae sinensis, active components purified therefrom, and the
derivatives according to the present invention may also be administered
as an adjuvant in combination with chemotherapy drugs, such as
actinomycin, adriamycin, Ara-C, bleomycin, carmustin, cisplatin,
cyclophosphamide, daunomycin, mitomycin, taxol, vinblastine, etc.

[0044]The acetone extract, chloroform extract or hexane extract of
Angelicae sinensis, active components purified therefrom, and the
derivatives according to the present invention may also be formulated in
any dietary compositions by using any techniques known to those skilled
in the art.

[0045]The following Examples are given for the purpose of illustration
only and are not intended to limit the scope of the present invention.

[0046]Materials and Methods

[0047]Preparation of Angelicae Sinensis Extracts and Compounds

[0048]The roots of Angelicae sinensis (Oliv.) were supplied from
Chung-Yuan Co., Taipei, Taiwan and were identified by Professor Han-Ching
Lin. A voucher of herbarium specimen was deposited at the School of
Pharmacy, National Defense Medical Center. The dried and powdered
rhizomes of Angelicae sinensis (12 kg) were extracted 3 times with
acetone (24 L/time) to give an acetone extract called AS-A. AS-A was then
subjected to chloroform extraction 3 times (24 L/times). The latter
extracts were concentrated under reduced pressure to yield 31.67 g of
chloroform extract, called AS-C (from 100 g of acetone extract). A hexane
extract (AS-H) was obtained by extracting AS-A with hexanes.
n-Butylidenephthalide (BP) was purchased from Lancaster Synthesis Ltd.
(Newgate, Morecambe, UK) and used without further purification. E- and
Z-forms of BP were separated with column chromatography and characterized
with HPLC and NMR. They were dissolved in DMSO, incubated with shaking at
25° C. for 1 hour and stored at 4° C. before each in vitro
experiment.

[0053]Homogenization: The cells were lysed directly in a 6 well culture
plate by adding 1 ml of trizol reagent to a 3.5 cm diameter well, and
passing the cell lysate several times through a pipette.

[0054]Phase separation: The homogenized samples were incubated for 5
minutes at 15 to 30° C. to permit the complete dissociation of
nucleoprotein complexes. Then, 0.2 ml of chloroform (Riedel-de-haen) per
1 ml of trizol reagent was added. Sample tubes were capped securely. The
tubes were vigorously shaken by hand for 15 seconds and incubated at 15
to 30° C. for 2 to 3 minutes. The samples were centrifuged at no
more than 12,000×g for 15 minutes at 2 to 8° C. Following
centrifugation, the mixture separated into a lower red, phenol-chloroform
phase, an interphase, and a colorless upper aqueous phase. RNA remained
exclusively in the aqueous phase.

[0055]RNA precipitation: The aqueous phase was transferred to a fresh
tube. The RNA was precipitated from the aqueous phase by mixing with
isopropyl alcohol (Fluka). 0.5 ml of isopropyl alcohol was added for
every 1 ml of trizol reagent which was added for homogenization
initially. The samples were incubated at 15 to 30° C. for 10
minutes and centrifuged at no more than 12,000×g for 10 minutes at
2 to 8° C.

[0056]RNA wash: The supernatant was removed. The RNA pellet was washed
once with 75% ethanol, using at least 1 ml of 75% ethanol for every 1 ml
of trizol reagent, which was added for the homogenization initially. The
sample was mixed by vortexing and centrifuged at no more than
7,500×g for 5 minutes at 2 to 8° C.

[0057]Re-dissolving the RNA: The supernatant was removed, and then the RNA
pellet was dried. The RNA was dissolved in RNase-free water, and
incubated for 10 minutes at 55 to 60° C. The RNA can be stored at
-70° C.

[0060]The effects on cell viability after the treatments with different
concentrations of the Angelicae sinensis extracts or the active
components purified therefrom were evaluated by modified MTT assay in
triplicate. Briefly, the cells (5×103) were incubated into
96-well plates containing 100 μl of a growth medium. The cells were
permitted to adhere for 24 hours, then treated with 100 μl of the
herbal extracts or the active components dissolved in the medium. The
control contained DMSO of ≦0.02% (v/v). After 24, 48 and 72 h
incubation, the drug-containing medium was replaced by 50 μl of fresh
medium, and cells in each well were incubated in 50 μl of 400 μg/ml
MTT for 6-8 h. The medium and MTT were removed later and 100 μl of
DMSO was added to each well and to the control, to dissolve the soluble
components. Absorbance at 550 nm of the solutions was measured with MRX
Microtiter Plate Luminometer (DYNEX, USA). The absorbance of untreated
cells was considered as 100%. To evaluate the effects of the extracts or
the active components on cell growth rate of GBM cells, 5×103
exponentially growing cells were treated with different concentrations
for 24, 48, or 72 h. The cytotoxicity of each test substance was
determined as an IC50 value, which represents the drug concentration
required to cause 50% inhibition. All experiments in this study were
performed in triplicate.

[0064]The inhibition effects of carmustin (BCNU) and Taxol were also
tested and compared. The results showed that GBM tumors were not
sensitive to carmustin (IC50>100 jig/ml) but DBTRG-05MG and
G5T/VGH GBM cells were sensitive to Taxol (IC50=61.0±3.3 μg/ml
and IC50<0.1 μg/ml, respectively). However, Taxol induced a
very high cytotocixity (IC50<0.1 μg/ml), which is much greater
than that induced by AS-C and BP, in vascular endothelia cells. After
treatment with AS-C or BP, the GBM cells (DBTRG-05MG) were seen to be
detached and floating in the media at different points of time within a
72-hour period of observation. The extent of GBM cell detachment and
flotation was found to increase with time, and with the increase in
dosage (in the case of BP when an observation was made at 3 hours).

[0065]The GBM cell detachment and flotation after treatment with AS-C or
BP, can be attributed to morphology change in the tumor cells. In the
above experiment, BCNU (carmustin) was used as the system control.

EXAMPLE 2

AS-C and BP Enhance the Cell Cycle Arrest at G0/G1 Phase in GBM Cells

[0066]Brain tumor cell lines DBTRG-05MG and G5T/VGH were cultured in the
growth medium with a diluent. For each test and control, DMSO was added,
and the content is less than 0.02% (v/v). For the AS-C and BP treatment,
70 μg/ml of AS-C and 400 μM of BP were added, respectively. All
were cultured for 48 hours. The analysis of cell cycle distribution was
performed by DNA staining with propidium iodide (PI). Briefly,
2×106 adherent cells were detached by trypsinization. The detached
cells and the floating dead cells were centrifuged and washed twice with
10 ml of cold 1×PBS (Life Technologies, Inc.). Supernatant was
aspirated, cells were re-suspended in 0.8 ml of 1×PBS, and then 200
μl of PI solution (50 μg/ml PI+0.05 mg/ml RNase A; Sigma Chemical
Co.) was added, and the cells were refrigerated at 4° C.
overnight. The cells were incubated while protected from light at room
temperature for at least 2 h before DNA analysis. After staining, DNA was
detected and quantified on 20,000 total cells using a FACScan (Becton
Dickinson Immunocytometry Systems, San Jose, Calif., USA) and CellQuest
analysis software. The G0/G1 phases were gated in M1 (×2); G2/M
phases were gated in M2 (×2); the total cells were gated in M3; S
phase was M3-(M1(×2)+M2(×2)); Sub G1 phase (apoptosis cells)
was gated in M4.

[0067]The cell cycle analysis demonstrated that both 70 μg/ml of AS-C
and 400 μM of BP enhanced the cell cycle accumulation at G0/G1 phase
(>90%) in GBM cells. FIGS. 1a and 1b showed that both AS-C and BP
enhanced a significant G0/G1 phase arrest with a concurrent decrease of S
phase after treatment for 12 hours to 48 hours p<0.05, p<0.005).

EXAMPLE 3

AS-C and BP Induce GBM Cells Apoptosis

[0068]Apoptotic cell death was analyzed using In Situ Cell Death Detection
Kit, POD (Roche, Germany). Changes in DNA chromatin morphologic features
were used for quantification. The procedures were performed in accordance
with the manufacturer's instructions. Briefly, cells were cultured on
culture dish and analyzed 72 hours after treatment with AS-C (70
μg/ml) and BP (5˜800 μg/ml), respectively. In AS-C and
BP-treated groups, the suspended cells were collected. In the control
group, adherent and floating cells were collected. Then, the cells were
fixed with 3.7% formaldehyde at room temperature for 15 min. on saline
coated slides, washed once in 1×PBS, and incubated in cold
permeabilization solution (0.1% Triton X-100+0.1% sodium citrate) after
reducing activity of endogenous peroxidase with 3% H2O2. The
cells were washed with 1×PBS again, and incubated with terminal
deoxynucleiotidyl transferase (TdT)-mediated dUTP nicks labeling (TUNEL)
reaction mixture for 60 minutes at 37° C. Then, the cells were
washed with 1×PBS, counterstained with propidium iodide (PI) for
cell counting. For quantification of apoptosis, the results were viewed
under fluorescence microscopy (Nikon, Kawasaki, Japan).

[0069]When compared to untreated cells, almost all detached GBM cells in
the AS-C and BP-treated groups were found to have undergone apoptosis.
The apoptotic cells with AS-C treatment were detected by fluorescence
microscopy (400×), with In Situ TUNEL staining and propidium iodide
cell counterstaining, and observed under light field. Likewise, BP
induced apoptosis in GBM cells was assessed by TUNEL method and using
propidium iodide as a counter stain. The GBM cells were exposed to BP (5
to 800 μM) for 48 hours before assessment. The results were shown in
FIG. 2. It was found that as compared to the untreated cells (control),
the apoptosis rates of GBM cells in the BP treatment group, were much
higher.

EXAMPLE 4

AS-C and BP Induce Apoptosis through the Activation of Multiple Pathways

[0072]The phosphorylations of p53 and Rb proteins were monitored and the
results showed the AS-C increased phosphorylated p53 protein at 6 h after
treatment. Furthermore, the amount of total p53 protein was increased as
well at 6 h and then gradually decreased. However, phosphorylated Rb
protein was seen to decrease at 6 h, and became undetectable at 12 h
after AS-C treatment. These results indicated that AS-C could trigger the
cell cycle checkpoint machinery. The amounts of pl6, p21 and Bax in AS-C
treated GBM cells were consequently measured and all of these three
proteins were found to increase after treatment with AS-C (see FIG. 3b).

[0073]Finally, the activations of procaspase-9 and procaspase-3 were also
determined. Both procaspase-9 and procaspase-3 were highly activated at 6
h after AS-C treatment (FIG. 3c).

[0074]In the case of BP, the results showed that BP 400 μM greatly
increased the expression of Fas (from 5.2 times at 1.5 h to 27.9 times at
48 h), while suppressing the expression of the Fas Ligand on the GBM
cells (see FIG. 3d).

[0075]It is also observed that BP enhanced the activation of caspase 8,
which was increased to 137.9 by the 48 h, while procaspase 8 declined
(see FIG. 3d).

[0076]The study of the role of mitochondrial pathway in BP-induced
apoptosis showed that BP induced Bax and AIF expression, which increased
to 16 times and 2.4 times respectively, by the 48 h, and activated
caspase-9 by 25.8 times at the 48 h while procaspase-9 declined (see FIG.
3e). Caspase-3 was also observed to increase while procaspase-3 declined
(FIG. 3d).

[0077]The study of the role of cell cycle pathway in BP-induced apoptosis
showed that BP increased p53, p21 and p16 expression by 1.4, 2.3 and 3.1
times respectively at 48 h. It also increased p53 phosphorylation by 5.2
times at 1.5 h and 9.2 times at 48 h, but decreased Rb phosphorylation by
0.2 times at 48 h (FIG. 3f). Beta-actin was used as an internal control
in this study. In FIG. 3g, BP was seen to decrease also cdk2, cdk4, cdk6
cyclins DI and cycline E.

[0078]In conclusion, we theorize a schematic model of the apoptosis signal
transduction pathways induced by BP stress, which consists of death
receptor, mitochondrial and cell cycle pathways.

EXAMPLE 5

Animal Studies

[0079]The RG2 cells (rat GBM) and DBTRG-05MG cells (human GBM) were used
in animal experiments to monitor the anti-tumor activities of AS-C and
BP. Male F344 rat (230-260 g) and male Foxn1 nu/nu mice (10-12 weeks)
were obtained from National Laboratory Animal Center (Taipei, Taiwan).
All procedures were performed in compliance with the Standard Operation
Procedures of the Laboratory Animal Center of Tzu Chi University
(Hualien, Taiwan). Animals were kept under pathogen-free conditions and
fed a standard laboratory diet. The DBTRG-05MG cells (human GBM) and RG2
cells (rat GBM) were prepared for nude mice xenografts and rat
allogenics, respectively.

[0081]Syngeneic F344 rats in two groups (6/group) were implanted
subcutaneously on the back with 1×106 RG2 cells. The animals
were administered by subcutaneous injection either with AS-C (500
mg/kg/day) (treatment group), or with the vehicle (50 mg/ml propylene
glycol and 100 mg/ml Tween-80 in distilled water; Standard Chem. &
Pharm., Tainan, Taiwan) (control group), at a spot distant from the
inoculated tumor sites (>2 cm), on day 3, 6 and 9, after tumor cell
implantation. Tumor sizes were measured using a caliper and the volume
was calculated as L×H×W×0.52. The animals were
sacrificed when tumor volume exceeded 25 cm3 and the day of
sacrifice was assumed as the final survival day for the animals.

[0082]The results showed that AS-C treatment had a significant inhibitory
effect on tumor growth when compared with the untreated (control) group
(p<0.05) (FIG. 4). The average tumor size at day 26 was 20.7±1.5
cm3 for the control group and 11.5±0.7 cm3 for the treatment
group, respectively. Survival of rats in the AS-C treated group was
significantly prolonged, compared with those in the control group
(40±2.7 days vs. 30±2.1; p<0.0001) (FIG. 5).

[0083]With a dose of 500 mg/kg subcutaneous injection of AS-C, no drug
related toxicities were observed in the rats as evidenced by the results
of the body weights and histological analysis of the vital organs.

[0085]Nude mice, in two groups (6/group), were implanted s.c. with
5×106 DBTRG-05MG cells, and administered with AS-C (i.p. 500
mg/kg/day), AS-C (s.c. 500 mg/kg/day) or vehicle (s.c.) on day 5 after
tumor cell implantation. Tumor sizes were measured using a caliper and
the volume was calculated as L×H×W×0.52. The animals
were sacrificed when tumor volume exceeded 1000 mm3 in mice, and the
day of sacrifice was assumed as the final survival day for the animals.

[0086]The results showed that there were significant suppressions of tumor
growth in the AS-C i.p. (500 mg/kg) and AS-C s.c. (500 mg/kg) treated
groups compared with the untreated group Up <0.005). The mean values
of tumor sizes at day 38 were 849.9±150.1 mm3 in the control
group, 295.5±25.3 mm3 in AS-C i.p. (500 mg/kg) treated group and
155.1±56.4 mm3 in AS-C s.c. (500 mg/kg) treated group. The
results were shown in FIG. 7.

[0088]The cytotoxic effect of AS-C on in situ tumor was determined with
RG2 cells. Syngeneic rats in two groups (6/group), were implanted i.c.
(striatum) with 5×104 RG2 cells, and treated with AS-C (500
mg/kg/day) or vehicle s.c. at day 4, 5, 6, 7 and 8 after tumor cell
implantation. Tumor volumes were measured and calculated by 3-T unit MRI
(General Electric, Wisconsin, USA) with echo-planar imaging capability
(Signa LX 3.8, General Electric, Wisconsin, USA) in Buddhist Tzu Chi
General Hospital (Hualien, Taiwan). Briefly, rats were anesthetized with
chloral hydrate (400 mg/ml, 1 ml/100g). Functional MRI scanning was
conducted with a fast spin echo, echo-planar acquisition sequence in
which the repetition time was 6000 msec, the echo time was 102 msec, the
matrix image was 256 by 256, the field of view was 5 by 5 cm, and the
in-plane resolution was 80 μm. Twenty slices, each 1.5 mm thick, were
obtained every 19.5 seconds for 6.5 minutes for each rat.

[0089]Significant declines of tumor volume in the treated group were
observed in the MRI image data, compared with the untreated group
(p<0.05) (FIG. 6). The mean tumor volumes at day 14 and day 16 were
70±4.8 mm3 and 126.4±11.1 mm3 in the control group versus
46.2±3.6 mm3 and 99.5±9.5 mm3 in AS-C treated group.

[0090]Experiment 4--Cytotoxicities of AS-C administered by subcutaneous
injection, on the tumor size of mice bearing xenograft human GBM tumor In
this experiment, the tumor was allowed to grow to a larger size to
simulate a clinical condition where surgical removal of tumor was not an
acceptable option.

[0091]As described above, DBTRG-05 MG cells (5×106) were
implanted s.c. on the backs of nude mice. The tumor-bearing mice were
treated with single dose of AS-C (500 mg/kg) or vehicle (s.c.) only when
the tumor volumes were ≧250 mm3. The mice were sacrificed to
determine the cytotoxicities in tumors by H&E tissue staining at day 10
after treatment of AS-C. The tissue sections were observed and
photographed under a light microscope at magnifications of 50× and
400×.

[0092]The photographs of histology analysis showed that AS-C had induced a
nucleic degradation, a cavity cytosol and tumor cell death in the tumor
cell mass. In contrast, the control tumor were growing very well and the
cytotoxic effects as seen in the AS-C treated group, were not found in
the tumor mass.

[0095]The rats in two groups (6/group) were implanted i.c. (striatum) with
5×104 RG2 cells, and randomly treated with BP (300 mg/kg/day) or
vehicle s.c. in the hind flank region after tumor cell implantation at
day 4, 5, 6, 7 and 8 for five dosages. Tumor volume was measured and
calculated by MRI. MRI was performed with a 3-T unit (General Electric,
Wisconsin, USA) with echo-planar imaging capability (Signa LX 3.8,
General Electric, Wisconsin, USA). Briefly, rats were anesthetized with
chloral hydrate (400 mg/ml, 1 ml/100 g). Functional MRI scanning was
conducted with a fast spin echo, echo-planar acquisition sequence in
which the repetition time was 6000 msec, the echo time was 102 msec, the
matrix image was 256 by 256, the field of view was 5 by 5 cm, and the
in-plane resolution was 80 μm. Twenty slices (1.5 mm thick each) were
obtained every 19.5 seconds for 6.5 minutes for each rat. Finally, the
whole tumor sizes (mm3) were measured and calculated for each group.

[0096]The results indicated that there was a significant reduction of
tumor volume in the BP treated group as compared with the untreated group
(p<0.05), as calculated using MRI scanning and Echo-planar as
described above (FIG. 8). Each column in FIG. 8 represents a mean±SE
(*:p<0.05; **: p<0.001). The mean of tumor volumes at day 14 and
day 16 were 69.9±4.81 mm3 and 126.43±11.07 mm3 for the
control group respectively; and 46.6±1.8 mm3 and 91.68±8.3
mm3 for the BP-treated group respectively. MRI image data showed
that the tumor volume in situ of BP-treated group had a smaller region
than control group.

[0097]Experiment 2: Effects of BP on suppressing the growth of s.c.
xenograft human GBM tumors in nude mice, and on the survival rate of the
mice.

[0098]Animals (Foxn1 nude mice) in six groups (6/group) were implanted
subcutaneously (s.c.) with 1×106 DBTRG-05MG cells, and
randomly treated with BP s.c. (70, 150, 300, 500, 800 mg/kg/day) or
vehicle s.c. at a site remote (>2 cm) from the incubated tumor after
tumor cell implantation, at day 4, 5, 6, 7 and 8 for all five dosages.
Tumor size was measured every 2 days and tumor volume was calculated.
Animals were sacrificed when tumor exceeded 1000 mm3. Tumor growth
was monitored for 3 months for those not sacrificed. Survival rate was
followed for up to 200 days.

[0099]The results showed that there were significant suppressions of tumor
growth in the BP-treated groups compared with the untreated group (FIG.
9; p<0.005 for the 300 mg/kg group), and the degree of tumor growth
inhibition is dose dependent.

[0100]Log-rank (Mantel-Cox) comparison of survival plots given in FIG. 10
indicated that the survival period of nude mice with xenograft
subcutaneous human GBM was prolonged up to 200 days upon treatment with
BP.

[0104]Data were expressed as the mean±SD or SE. Statistical
significance was analyzed by Student's t-test. A p value of <0.05 was
considered significant. Survival was compared by log-rank (Mantel-Cox)
test. Median survival time was estimated from Kaplan-Meier analysis.

[0105]While embodiments of the present invention have been illustrated and
described, various modifications and improvements can be made by persons
skilled in the art. It is intended that the present invention is not
limited to the particular forms as illustrated, and that all the
modifications not departing from the spirit and scope of the present
invention are within the scope as defined in the appended claims.